Histone deacetylase (hdac) inhibitor up-regulates car expression and targeted antigen intensity, increasing antitumor efficacy

ABSTRACT

Embodiments of the invention employ methods and compositions for enhancing potency of immune cells that express one or more therapeutic proteins. In certain cases, the methods modulate expression of a CAR transgene in an immune cell, such as a T cell. Specific embodiments employ the exposure of cells and/or individuals to be treated with the cells with an effective amount of at least one agent that upregulates expression of the therapeutic protein, such as a mitogen, histone deacetylase inhibitor, and or DNA methyltransferase inhibitor.

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/847,957, filed Jul. 18, 2013, which application isincorporated by reference herein in its entirety.

This invention was made with government support under W81XWH-11-1-0625awarded by the Department of Defense Prostate Cancer Research Programand CA094237 and P50 CA058183 awarded by the NIH. The government hascertain rights in the invention.

TECHNICAL FIELD

The field of the present invention includes at least the fields of cellbiology, molecular biology, immunology, and/or medicine, includingcancer medicine.

BACKGROUND

T cells modified to express tumor-directed chimeric antigen receptors(CARs) have shown clinical efficacy in treating both hematologicalmalignancies and solid tumors. It is likely, however, that the mosteffective use of CAR-modified T cells will require additionalengineering to enable them to overcome tumor immune escape mechanisms.One of these escape strategies is target antigen modulation underselective pressure. This phenomenon has been reported as a cause offailure in both preclinical and clinical studies usingadoptively-transferred T cells with single antigen specificity to treatheterogeneous tumors.

SUMMARY

The methods and compositions provided herein address the potentialproblem of target antigen modulation, e.g., reduction of target cancerantigen expression, under selective pressure, e.g., during chemotherapy,by providing methods and compositions effective to circumvent suchmodulation. The methods and compositions herein utilize one or moreagents that upregulate expression of a therapeutic protein, e.g., achimeric antigen receptor (CAR), in an immune cell, e.g., T lymphocyte,such that the cell maintains efficacy as a therapeutic even when targetantigen expression is reduced by selective pressure. In certainembodiments, the expression of the therapeutic protein is upregulated bythe agent in a sufficient amount to allow the immune cell to be orremain therapeutically effective. In specific embodiments, theexpression is upregulated in the cell by the agent to a level that isgreater than that in a cell that is not exposed to the agent.

In one aspect, provided herein is a method of enhancing potency, e.g.,cancer cell-killing activity, of immune cells that express at least onetherapeutic protein, by exposing one or more agents to such immune cellssuch that expression of said at least one therapeutic protein therein isupregulated or increased compared to such immune cells that are notexposed to the one or more agents. In certain embodiments, providedherein is a method of increasing the cancer cell-killing activity of apopulation of immune cells that express a therapeutic protein, e.g., aCAR, comprising contacting the immune cells with one or more agents fora time and in an amount sufficient to increase expression of thetherapeutic protein. In certain embodiments, provided herein is a methodof increasing cancer cell-killing activity of a population of immunecells that express a therapeutic protein, e.g., a CAR, comprisingcontacting the immune cells with one or more agents for a time and in anamount sufficient to increase expression of the therapeutic protein andthereby increase the cancer cell-killing activity of the population ofimmune cells. In specific embodiments, the upregulation or increase inexpression is detectable by standard means, such as by western blottingor Northern blotting, for example.

In particular embodiments, the population of immune cells is contactedwith one or more agents to upregulate expression of the therapeuticprotein when the cells are in vitro, ex vivo, and/or in vivo. In oneembodiment, the population of immune cells is contacted with the one ormore agents an individual who has been administered, or is to beadministered, the immune cells. In specific embodiments, the immunecells are contacted with the one or more agents prior to administrationof the immune cells to the individual, or the immune cells are contactedwith the one or more agents after administration of the immune cells tothe individual. In a specific embodiment, the method comprises isolatingimmune cells lacking the therapeutic protein from an individual;engineering the immune cells to express the therapeutic protein;administering the engineered immune cells to the same individual; andsubsequently administering the agent to the individual in an amountsufficient to cause an increase in the expression of the therapeuticprotein in the immune cells. Immune cells from the individual may beobtained at or near the time of therapy or may have been suitably storedprior to therapy.

In certain embodiments, therapeutic protein-expressing immune cells tobe delivered to an individual are obtained from one or more otherindividuals. Such cells may be assayed and/or manipulated such that theyare safe to be delivered to another individual and are less likely to berejected by the recipient individual's own immune system, for example.The cells may be stored in a repository, for example.

In specific embodiments of any of the embodiments herein, the agentcomprises one or more of an epigenetic modifier and/or a mitogen. Inparticular embodiments, the epigenetic modifier is a histone deacetylase(HDAC) inhibitor, DNA methyltransferase (DNMT) inhibitor, or combinationthereof. When more than one agent is employed to upregulate expression,any combination may be employed. The combination may encompass two ormore of the same type of agent or two or more different types of agents.For example, the cells may be exposed to two types of epigeneticmodifiers or one type of epigenetic modifier and a mitogen. When morethan one epigenetic modifier is employed, the combination may includetwo HDAC inhibitors or one HDAC and one DNMT inhibitor, for example.

In one embodiment, provided herein is a method of enhancing potency ofimmune cells that express at least one therapeutic protein, comprisingcontacting the immune cells with an effective amount of a mitogen,histone deacetylase (HDAC) inhibitor, and/or deoxyribonucleic acidmethyl transferase (DNMT) inhibitor for a time sufficient for expressionof said therapeutic protein to increase, as compared to said immunecells not contacted with said mitogen, HDAC inhibitor and/or said DNMTinhibitor. in specific embodiments, the HDAC inhibitor a small chainfatty acid, hyroxamic acid, cyclic peptide, benzamide or a combinationthereof. In particular embodiments, the HDAC inhibitor is one or more oftrichostatin A, sodium phenylbutyrate, Buphenyl, Ammonaps, Depakote,valproic acid, romidepsin (ISTODAX®), Vorinostat, Zolinza, panobinostat,belinostat, entinostat, JNJ-26481585, MGCD-010, and/or a combinationthereof. In other specific embodiments, the DNMT inhibitor is anucleoside analog, quinolone, active site inhibitor, or a combination ofany thereof. Specific DNMT inhibitors may be selected from the groupconsisting of 5-azacitidine, decitabine, zebularine, SGI-110, SGI-1036,RG108, caffeic acid purum, chlorogenic acid, epigallocatechin galiate,procainamide hydrochloride, MG98, and a combination thereof. In specificcases, the DNMT inhibitor is a ribonucleoside analog ordeoxyribonucleoside analog. The DNMT inhibitor may be decitabine orzebularine. The DNMT inhibitor may be 5-azacytidine, such as VIDAZA®.

In embodiments, the immune cells are T cells (including CD4+ T cells,CD8+ T cells, or Treg cells), NK cells, dendritic cells, or a mixture ofany thereof.

In some cases, contacting of the immune cells and the mitogen, HDACinhibitor, and/or DNMT inhibitor is performed in vitro and thecontacting may occur in cell culture. In some embodiments, thecontacting is performed in a pharmaceutical composition comprising theimmune cells and the HDAC inhibitor and/or the DNMT inhibitor. Inparticular aspects, the contacting is performed in vivo, and the immunecells are T cells in an individual.

In particular aspects, immune cells and the mitogen, HDAC inhibitor orthe DNMT inhibitor are administered to an individual separately, such asin separate pharmaceutical formulations. In certain cases, the mitogen,HDAC inhibitor and/or DNMT inhibitor, and the immune cells areadministered to the individual in the same pharmaceutical formulation.The mitogen, HDAC inhibitor and/or the DNMT inhibitor, and the immunecells, may be administered to the individual at substantially the sametime or at different times.

The mitogen, HDAC inhibitor and/or the DNMT inhibitor, and the immunecells, may be administered to an individual according to the same dosingschedule or according to different dosing schedules. In particularaspects, the mitogen, HDAC inhibitor, and/or DNMT inhibitor are providedto the individual before the individual receives immune cells. In someaspects, the immune cells are contacted with the mitogen, HDACinhibitor, and/or DNMT inhibitor prior to being delivered to theindividual. The immune cells may not be contacted with the mitogen, HDACinhibitor, and/or DNMT inhibitor prior to being delivered to theindividual.

In some cases, the immune cells are contacted with at least one HDACinhibitor and at least one DNMT inhibitor. In particular embodiments,the immune cells are contacted with said HDAC inhibitor in vitro, andsubsequently contacted with said DNMT inhibitor in vivo. The immunecells may be contacted with the DNMT inhibitor in vitro, andsubsequently contacted with said HDAC inhibitor in vivo. The expressionof the therapeutic protein in the immune cells may be controlled by apromoter at least a portion of the sequence of which is methylated, andwherein the methylation results in partial or complete silencing ofexpression of said therapeutic protein. The expression of thetherapeutic protein in the immune cells may be controlled by a promoterrepressor region, at least a portion of the sequence of which ismethylated, and wherein the methylation results in enhanced expressionof said therapeutic protein.

Immune cells may be contacted with two or more HDAC inhibitors, with twoor more DNMT inhibitors, or with two or more mitogens. In some cases,the immune cells are contacted with an HDAC inhibitor and a DNMTinhibitor or an HDAC inhibitor and a mitogen, or a DNMT inhibitor and amitogen.

In certain embodiments, the contacting step occurs multiple times invivo in the individual. In particular aspects, in a second or subsequentcontacting step, the immune cells express a different therapeuticprotein than immune cells in a first contacting step. In certain cases,in a second or subsequent contacting step, a mitogen, HDAC inhibitorand/or DNMT inhibitor is administered to the individual. The mitogen,HDAC inhibitor, and/or DNMT inhibitor in the second or subsequentcontacting step may be different or the same than the mitogen, HDACinhibitor, and/or DNMT inhibitor in the first contacting step.

In embodiments wherein more than one epigenetic modifier and/or mitogenare exposed to immune cells expressing one or more therapeutic proteins,the multiple agents may be exposed to the cells in any suitablechronology, dosing schedule, and/or formulation(s). In specificembodiments, the two or more agents are provided to an individual prior,during, and/or after delivery of the immune cells and are provided atthe same time, although in some cases they are provided at differenttimes. When multiple agents are delivered to an individual, they may becomprised in the same or different formulations, and they may bedelivered by the same or different routes to the individual when theyare not in the same formulation. When multiple agents are provided toimmune cells and/or an individual, the agents may have the same ordifferent dosing schedule and/or doses.

In specific embodiments, the therapeutic protein expressed by the immunecells is a receptor. In specific cases, the receptor targets an antigenon a cancer cell. The therapeutic protein may be of any kind, such as achimeric antigen receptor (CAR), cytokine, cytokine receptor, ligandtrap, antibody (including a monomeric or multimeric antibody), anengineered αβT cell receptor, or an antigen-specific receptor. In caseswhere an immune cell expresses more than one therapeutic protein, thetherapeutic proteins may be of the same type (both CARs, for example) ormay be of different types (CAR and an engineered αβT cell receptor).When the immune cells express more than one therapeutic protein, theymay both be upregulated in expression upon exposure to the one or moreagents.

The therapeutic protein may be of any kind, but in specific embodiments,it is a chimeric antigen receptor (CAR). The CAR may comprise at leastone extracellular antigen-binding domain and at least one intracellularsignaling domain. In certain aspects, the therapeutic protein is acytokine, cytokine receptor, or ligand trap. The therapeutic protein maybe a monomeric or multimeric antibody. The therapeutic protein may be aαβT cell receptor or an antigen-specific receptor. In specificembodiment, the immune cells are autologous or allogeneic to aparticular individual. The therapeutic protein may be a transgenicprotein.

Immune cells may comprise two or more different therapeutic proteins. Insome cases, the two different therapeutic proteins are both receptors.In particular embodiments, the two different therapeutic proteins areboth CARs. In some cases, one of the two different therapeutic proteinsis a CAR and one of the two different therapeutic proteins is anantibody or one of the two different therapeutic proteins is a CAR andone of the two different therapeutic proteins is an αβT cell receptor,or one of the two different therapeutic proteins is a CAR and one of thetwo different therapeutic proteins is an antigen-specific receptor.

In specific embodiments, there are immune cells that comprise more thanone CAR and/or that comprise one or more CARs that target at least twoantigens. The target antigens may be cancer antigens of any kind. Inspecific embodiments the CARs target MUC1 and PSCA, both of which areexpressed on approximately 60% of human primary pancreatic cancer cells,as an example. As described herein, there is characterization in anexemplary embodiment of a pancreatic tumor model whether immune escapecould be prevented by co-administering CAR-T cells targeting twoantigens present on the tumor cells. As expected, when testedindividually, selective pressure resulted in the emergence of a tumorsubpopulation that lacked or had downregulated the target antigen,rendering the tumor insensitive to subsequent T cell retreatment.Unexpectedly, however, it was determined that the co-administration ofCAR-T cells simultaneously targeting both TAAs, though associated withsuperior anti-tumor effects, was also insufficient to produce tumorelimination.

When the immune cells are modified or engineered to express more thanone therapeutic protein, the therapeutic proteins may be delivered tothe immune cells in the form of nucleic acids capable of expressing thetherapeutic proteins. Such nucleic acids may be expression constructscomprising regulatory sequences that control expression of thetherapeutic protein(s). In cases wherein expression construct(s) aredelivered to the immune cells such that more than one therapeuticprotein is to be expressed by the immune cells, the therapeutic proteinsmay or may not be encoded from the same expression construct. Inspecific embodiments of the expression construct, there may be more thanone regulatory sequence that regulates expression of the therapeuticprotein. In specific embodiments, the regulatory sequences may be apromoter. In particular embodiments, at least a portion of the promoteris methylatable. In specific embodiments, at least a portion of thepromoter is methylated. In particular embodiments, methylation of thepromoter results in partial or complete silencing of expression of thetherapeutic protein. In specific embodiments, the promoter comprises oneor more CpG islands.

In certain embodiments, a particular individual is treated with morethan one exposure to immune cells comprising at least one therapeuticprotein; that is, the individual is administered a dose of the immunecells two or more times during the course of therapy. In second or moreexposures to therapeutic-protein comprising immune cells, the individualmay be provided immune cells expressing the same therapeutic protein(s).In certain cases, however, the individual may be provided immune cellsexpressing different therapeutic proteins. In cases wherein anindividual is provided two or more exposures to the cells, theindividual and/or the cells are also again contacted with one or moreagents that upregulate expression of the therapeutic protein. In caseswhere the second or subsequent rounds have immune cells with differenttherapeutic proteins compared to the initial round of immune cells, thedifferent therapeutic proteins may or may not target the same antigen.In cases where the second or subsequent immune cells comprise atherapeutic protein that targets a different antigen, the second orsubsequent antigen may reside on cancer cells that also expressed thefirst targeted antigen. For example, pancreatic cancer cells oftencomprise the tumor associated antigens (TAA) mucin1 (MUC1) and prostatestem cell antigen (PSCA), and the first set of immune cells may targetMUC1, whereas the second or a subsequent set of immune cells may targetPSCA (or vice versa).

For a particular target (e.g., tumor associated antigen or tumorspecific antigen), in certain embodiments, it is assumed that tumorescape will occur, and a second round of treatment with the immune cellsexpressing a therapeutic protein incorporates contacting the recipientwith the epigenetic modifier agent or mitogenic agent, e.g., in anamount that results in increased expression of the therapeutic protein.In other embodiments, upon the second round of treatment with the immunecells expressing a therapeutic protein, the recipient of the cells maybe monitored for evidence of tumor escape (e.g., by sampling tissue fromthe recipient and analyzing for one or more tumor markers (e.g., TAAs orTSAs), and administering the epigenetic modifier agent and/or mitogenicagent upon evidence of tumor escape (e.g., recurrence).

In some embodiments, the immune cells encompass an inducible suicidegene, such as an inducible caspase, e.g., caspase-9, e.g., iCaspase9,Herpes Simplex Virus (HSV) thymidine kinase (TK) gene, cytosinedaminase, purine nucleoside phosphorylase, diphtheria toxin, andnitroreductase.

In particular embodiments, an individual is provided an additionaltherapy in addition to the immune cells/agent of the disclosure. Forexample, an individual with cancer may receive the immune cells/agenttherapy as encompassed herein and additionally receives an additionaltherapy, e.g., one or more of chemotherapy chemotherapy (e.g., with anagent that is not a mitogenic agent or an epigenetic modifier agent),radiation therapy, hormone therapy, immunotherapy, and surgery. Theindividual may be administered the additional therapy before, during, orafter, or a combination thereof, administration of the immune cells andagent therapy.

In some embodiments, the methods comprise diagnostic steps wherein anindividual is diagnosed with cancer, such as using standard means in theart, including biopsy, cancer marker analysis, histology, and so forth,for example. In some embodiments, an individual is suspected of havingcancer or has the recurrence of cancer and is provided the therapydescribed herein. In certain embodiments, an individual is at risk ofhaving cancer or is at risk of having the return of cancer and isprovided the therapy described herein.

One embodiment provides a method of treating and/or preventing cancer inan individual, comprising administering to the individual the immunecells and agents that upregulate expression of therapeutic proteins inan amount effective to treat and/or prevent cancer in the individual.The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof cancer in a mammal. Furthermore, the treatment or prevention providedby the inventive method can include treatment or prevention of one ormore conditions or symptoms of the disease, e.g., cancer, being treatedor prevented. Also, for purposes herein, “prevention” can encompassdelaying the onset of the disease, or a symptom or condition thereof.

The individual referred to herein can be any individual. The individualmay be a mammal, including a human, dog, cat, horse, and so forth.

In any of the embodiments herein, the cancer is primary cancer ormetastatic cancer. The cancer may be, e.g., a solid tumor or a bloodcancer. With respect to the inventive methods, the cancer can be anycancer, including any of acute lymphocytic cancer, acute myeloidleukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, braincancer, breast cancer, cancer of the anus, anal canal, or anorectum,cancer of the eye, cancer of the intrahepatic bile duct, cancer of thejoints, cancer of the neck, gallbladder, or pleura, cancer of the nose,nasal cavity, or middle ear, cancer of the oral cavity, cancer of thevulva, chronic lymphocytic leukemia, chronic myeloid cancer, coloncancer, esophageal cancer, cervical cancer, fibrosarcoma,gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer,kidney cancer, larynx cancer, leukemia, liquid tumors, liver cancer,lung cancer, lymphoma, malignant mesothelioma, mastocytoma, melanoma,multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, ovariancancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer,pharynx cancer, prostate cancer, rectal cancer, renal cancer, skincancer, small intestine cancer, soft tissue cancer, solid tumors,stomach cancer, testicular cancer, thyroid cancer, ureter cancer, andurinary bladder cancer.

DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present embodiments, referenceis made to the following descriptions taken in conjunction with theaccompanying drawings.

FIG. 1 shows that T cells can be engineered to recognize and killpancreatic cancer cells expressing PSCA. (a) Retroviral vector map of afirst generation humanized, codon-optimized CAR specific for PSCA, (b)left panel, a dot plot from a representative donor showing control (NT)and CAR-PSCA transgenic (CH2CH3 positive) cells; right panel, summarytransduction efficiency data for 10 donors, represented as mean±standarddeviation (SD) is shown, (c) Phenotype of NT and CAR-PSCA T cells, n=10,(d) 6-hr chromium release assay of CAR-PSCA and NT T cells using PSCA+(CAPAN1) and PSCA− (293T cells) as targets. Data represents the mean±SDtarget specific lysis at an E:T of 10:1 (n=5).

FIG. 2 demonstrates that targeting of a heterogeneous tumor withmonospecific CAR-T cells leads to tumor immune escape. To assess whetherCAR-PSCA could control CAPAN1 tumor growth in vivo SCID mice wereengrafted with tumor cells. (a, left panel) shows bioluminescence imagesof a representative untreated mouse (top) or an animal treated withCAR-PSCA T cells at pre or 28, 42 or 56 days post treatment, while theright panel shows summary data for 4 mice. Data is plotted as foldincrease of luminescence intensity compared with day 0 (mean±SD). Toassess, in vitro, whether CAR-PSCA T cells could eliminate CAPAN1 (GFP+)cells, a 72-hr co-culture experiment was performed at a 5:1 E:T ratio,with NT T cells serving as controls. b shows the percentage of residualtumor cells, as quantified using flow cytometry and gating on GFP+ cellsand results are reported as mean±SD (n=4). (c) To assess whether CAPAN1cells that were resistant to initial CAR-PSCA T cell treatment could bekilled upon subsequent re-treatment, a subsequent co-culture wasperformed using either CAPAN1 cells initially treated with NT T cells(left panel) or those treated with CAR-PSCA T cells (right panel) astargets (E:T 5:1). After 72-hours residual tumor cells were againquantified by flow. Data is reported as the mean±SD; n=4. (d) Todetermine the mechanism of tumor resistance to T cell treatment, IHCanalysis of CAPAN1 cells was performed only or post NT or CAR-PSCA Tcell treatment. After exposure to control NT T cells tumor cells expressboth MUC1 and PSCA, whereas the majority of tumor cells treated withCAR-PSCA T cells have lost or express only low levels of PSCA antigenwhile retaining expression of MUC1.

FIG. 3 demonstrates CAR-T cells modified to recognize MUC1 kill MUC1+tumor cells. (a) shows a retroviral vector map of a first generationMUC1-specific CAR co-expressing a truncated form of CD19 (ΔCD19), b(left panel) shows a dot plot show representing the percentage oftransgenic (CH2CH3 and ΔCD19 double-positive cells) in a representativedonor, while the right panel shows summary data for 7 donors, (mean±SD).c shows the T cell phenotype of NT and CAR-MUC1 T cells (n=7). d showsin a 6-hr cytotoxicity assay, that CAR-MUC1 T cells can specificallykill MUC1+ targets (CAPAN1) with no recognition of control (293T)targets. NT T cells were used as additional controls and the datapresented shows % specific lysis at an E:T of 10:1 (mean±SD) for 5donors.

FIG. 4 provides that targeting a heterogeneous tumor with MUC1-specificCAR-T cells also leads to tumor immune escape. (a, left panel) showsbioluminescence images of representative mice engrafted withCAPAN-eGFP-FFluc intraperitoneally and administered either NT (top) orCAR-T cells (bottom). Imaging was performed prior to T cell infusion andat pre or 28, 42 or 56 days post-treatment. (A, right panel) showssummary data for 4 mice, with fold increase of luminescence intensitycompared with day 0 plotted, (mean±SD). b shows that in an in vitro72-hour co-culture assay not all CAPAN1/GFP-expressing cells are killed(n=3). Residual tumor cells were quantified by flow cytometry and gatingon GFP and results are reported as mean residual tumor cells±SD; (c)shows that while tumor cells originally treated with NT T cells aresensitive to retreatment with CAR-MUC1 T cells, those that wereresistant to CAR-MUC1 T cells originally retained this resistance uponretreatment. Results represent data from a single donor using an E:Tratio of 5:1. (d) IHC of CAPAN1 cells treated with CAR-MUC1 T cellsshows weak/intermittent MUC1 staining with no impact on the PSCApositive population.

FIG. 5 shows that targeting tumors using a dual CAR approach producessuperior antitumor activity. To determine whether combination therapywith CAR-MUC1 and CAR-PSCA T cells would produce superior anti-tumoreffects, (a) a 6-hr cytotoxicity assays of NT, CAR-MUC1, CAR-PSCA or thecombination with CAPAN1 cells as targets was performed. Results areexpressed as % specific lysis±SD at an E:T of 10:1 (n=5). Whereindicated *, the P value was less than 0.05 using student t test. (b)also performed was a 72-hour co-culture study with the same panel ofeffectors and targets (E:T of 5:1, n=5). Residual tumor cells werequantified by gating on GFP+ cells and results are expressed as %residual tumor cells±SD. It was next assessed whether the combination ofCAR-MUC1 and CAR-PSCA T cells could control CAPAN1 tumor growth in vivoin SCID mice. (c) shows bioluminescence images of representative miceengrafted with CAPAN-eGFP-FFluc and treated with NT, CAR-MUC1, CAR-PSCAor the combination, while (d) shows summary results for the NT anddual-targeted groups (n=4) at pre or 28, 42, 56 or 63 days posttreatment. Data is plotted as fold increase of luminescence intensitycompared with day 0, (mean±SD).

FIG. 6 demonstrates characterizing the tumor immune escape phenomenonusing an artificial tumor model. a shows two retroviral vector maps, thefirst encoding the TAA MUC1, which has a variable number of proline-richsegments that are tandemly repeated (variable number tandem repeat;VNTR)³⁷ and co-expressing mOrange. The second encodes the TAA PSCA,which contains six transmembrane portions, and co-expressing GFP. bshows immunofluorescence staining for MUC1/mOrange and PSCA/GFP intransduced 293T cells, c shows a 72-hour co-culture experiment where amixture of 293T expressing MUC1/mOrange and 293T expressing PSACA/GFP(1:1 ratio) with incubated with either NT or CAR-PSCA T cells at an E:T10:1 (n=6). Residual tumor cells were quantified by gating on GFP+ andmOrange+ populations and results are expressed as % residual tumorcells±SD. (d) the frequency of detectable tumor cells are quantified atco-culture initiation (0), then at 12, 24, 36, 48 and 60 hours aftertreatment with CAR-PSCA T cells (n=6), and distinguished based onexpression of either GFP or mOrange. Results are expressed as % residualtumor cells. (e) To determine whether the intensity of antigenexpression correlated with sensitivity to T cell treated thefluorescence intensity of GFP was measured at co-culture initiation (0),then at 12, 24, 36, 48 and 60 hours after treatment with CAR-PSCA Tcells, n=6. Results are presented as maximum fluorescence intensity±SD,f shows a 72-hour co-culture experiment where a mixture of 293Texpressing MUC1/mOrange and 293T expressing PSACA/GFP (1:1 ratio) withincubated with either NT or CAR-MUC1 T cells at an E:T 10:1 (n=5).Residual tumor cells were quantified by gating on GFP+ and mOrange+populations and results are expressed as % residual tumor cells±SD. (g)Tumor cells are quantified over time based on expression of either GFPor mOrange (n=5). Results are expressed as % residual tumor cells. (h)To determine whether the intensity of antigen expression correlated withsensitivity to T cell treated the fluorescence intensity of mOrange wasmeasured at co-culture initiation (0), then at 12, 24, 36, 48 and 60hours after treatment with CAR-MUC1 T cells, n=5. Results are presentedas maximum fluorescence intensity±SD. (i) shows a 72-hour co-cultureexperiment where a mixture of 293T cells expressing MUC1/mOrange and293T expressing PSACA/GFP (1:1 ratio) were incubated with either NT orthe combination of CAR-MUC1 and CAR-PSCA T cells at an E:T 10:1 (n=5).Residual tumor cells were quantified by gating on GFP+ and mOrange+populations and results are expressed as % residual tumor cells±SD. (j)Sensitivity of the tumor cells to dual CAR therapy is assessed atinitiation (0), then at 12, 24, 36, 48 and 60 hours after treatment.Results are expressed as % residual tumor cells (n=5). (k) To determinewhether the intensity of antigen expression correlated with sensitivityto T cell treated the fluorescence intensity of mOrange was measured atinitiation (0), then at 12, 24, 36, 48 and 60 hours after treatment withthe combination of CAR-MUC1 and CAR-PSCA T cells, n=5. Results arepresented as maximum fluorescence intensity±SD.

FIG. 7 shows that tumor immune escape occurs irrespective of whether Tcells are modified with a 1^(st), 2^(nd) or 3^(rd) generation CARconstruct. (a) retroviral vector maps of 1^(st), 2^(nd) and 3^(rd)generation CAR-PSCA constructs and (b) representative data confirmingthat all 3 generations of CARs can be expressed on T cells, as measuredby flow cytometry (c) fold expansion of 1^(st), 2^(nd) and 3^(rd)generation CAR-PSCA modified T cells in a representative donor afterco-culture with PSCA-expressing K562 cells at 2:1 T cell:K562 cellratio. (d) T cells modified with 1^(st) 2^(nd) and 3^(rd) generationCARs show equivalent recognition of PSCA+ targets as assessed by a 6-hrcytotoxicity assay using 293T and CAPAN1 cells as targets (data ispresented as % specific lysis at an E:T 10:1 for one representativedonor as well as a 72-hour co-culture experiment performed at an E:T of5:1, n=1, (e) where residual tumor cells were quantified by flow, gatingon GFP+ cells.

FIG. 8 demonstrates that fluorescence intensity of mOrange wascorrelated with MUC1 antigen expression on the engineered 293T cellsexpressing MUC1-mOrange. a shows a dot plot of the control 293T cellsand the engineered 293T-MUC1-mOrange cells co-expressing MUC1 antigenand mOrange (MUC1 Ag and mOrange-double positive cells).

FIG. 9 shows that decitabine treatment upregulates MUC1 expression ofCAR-resistant T cells and re-sensitizes them to T cell treatment. Toassess whether CAPAN1 cells that were resistant to initial CAR-MUC1 Tcell treatment could be killed upon subsequent re-treatment we performeda 72-hr co-culture experiment at a 5:1 E:T ratio, with NT T cellsserving as controls. a (right panel) shows the percentage of residualtumor cells, as quantified using flow cytometry and gating on GFP+ cellsand results are reported as mean±SD (n=4) then a subsequent co-culture(left panel) using CAPAN1 cells initially treated with CAR-MUC1 T cellsas targets (E:T 5:1), after 72-hours residual tumor cells were againquantified by flow. Data is reported as the mean±SD; n=4. (b) Todetermine whether decitabine exposure would upregulate MUC1 expressionin this resistant population we evaluated antigen expression, by flow,on CAR-resistant cells (grey histogram) and those cultured in decitabine(black histogram). To next assess whether decitabine treatmentresensitized CAPAN1 cells to CAR-MUC1 T cells we performed a 72-hrco-culture experiment using CAPAN1 cells initially treated with CAR-MUC1T cells (c) then either left untreated or cultured with decitabine astargets (E:T 5:1). After 72-hours retreatment with CAR-MUC1 T cells,residual tumor cells were again quantified by flow. Data is reported asthe mean±SD; n=2.

FIG. 10 shows exemplary expression of a CAR-MUC1 on T cells.

FIG. 11 shows that higher expression of CAR on modified-T cells hasfaster killing kinetics (293T-MUC1-mOr dim (low MUC1 expression)).

FIG. 12 demonstrates that higher expression of CAR on modified-T cellshas faster killing kinetics. (293T-MUC1-mOr bright (high MUC1expression)).

FIG. 13 demonstrates that decitabine enhances CAR expression onmodified-T cells.

FIG. 14 shows that decitabine enhances antitumor effects of CAR-MUC1T-cells.

The foregoing has outlined rather broadly the features and technicaladvantages of the present subject matter in order that the detaileddescription of the methods provided herein that follows may be betterunderstood. Additional features and advantages of the methods providedherein will be described hereinafter which form the subject of theclaims. It should be appreciated by those skilled in the art that theconception and specific embodiments disclosed may be readily utilized asa basis for modifying or designing other structures for carrying out thesame purposes. It should also be realized by those skilled in the artthat such equivalent constructions do not depart from the spirit andscope of the subject matter as set forth in the appended claims. Thenovel features which are believed to be characteristic of the methodsprovided herein, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe methods provided herein.

DETAILED DESCRIPTION

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more. In specificembodiments, aspects of the subject matter may “consist essentially of”or “consist of” one or more elements or steps of the subject matter, forexample. Some embodiments of the subject matter may consist of orconsist essentially of one or more elements, method steps, and/ormethods of the subject matter. It is contemplated that any method orcomposition described herein can be implemented with respect to anyother method or composition described herein.

As used herein, the term “enhancing the potency of immune cells” isdefined as an increase or improvement in the biological properties(natural or recombinant in origin) of a given cell. These biologicalproperties can include, but are not restricted to (i) antigenspecificity, (ii) proliferation, (iii) migration, (iv) persistence,and/or (v) killing ability. Additionally, immune cells can be enhancedwith recombinant modifications such as (i) resistance to theimmunosuppressive tumor microenvironment, (ii) resistance to drugs,and/or (iii) suicide genes.

I. Immunotherapy Methods for Antitumor Activity

In various embodiments, immunotherapy methods for employing immune cellsfor antitumor activity are provided. Methods of killing tumor cells arecontemplated herein employing immune cells that express at least onetherapeutic protein. Methods comprise administering to an individual oneor more immune cells that have been contacted in vitro or in vivo withan agent that upregulates expression of a therapeutic protein in theimmune cell. In certain embodiments, the agent is a HDAC inhibitor, DNAmethyl transferase inhibitor, mitogen, or combination thereof. Inspecific embodiments, one or more target proteins on tumor cells arealso increased in expression upon exposure of the tumor cells to immunecells having upregulatd expression of the therapeutic protein(s).

In particular embodiments, particular dosing regimens to providecompositions of the invention to an individual in need of cancertreatment are provided. Such regimens include compositions forupregulation of expression of the therapeutic protein in the immune cellin addition to the cells themselves. For example, an agent thatfacilitates upregulation in expression of the therapeutic protein in animmune cell may be provided to the immue cell prior to delivery of thecell to the individual, and/or it may be provided to the cell subsequentto the delivery of the cell to the individual. The immune cell(s) andthe agent may be provided to the individual separately or together, andthey may or may not be in the same formulation, and they may or may notbe provided at the same time to the individual. In certain embodiments,more than one HDAC inhibitor, DNA methyl transferase inhibitor, and/ormitogen are exposed to the immune cells in vitro and/or in vivo, and theorder in which they are exposed to the immune cells may be of anysuitable kind so long as expression of at least one therapeutic proteinexpressed by the cells is increased.

In certain embodiments, an individual is treated for cancer by providingto the individual an effective amount of a combination of immune cellsexpressing one or more therapeutic proteins and one or more epigeneticmodulators and/or mitogenic agents. The treatment may utilize modulationof immune cells (such as T cells) from the individual, e.g., ex vivomodulation such that the immune cells express a therapeutic protein andthe cells are subsequently exposed to one or more epigenetic modifiersand/or mitogenic agents prior to and/or subsequent to delivery to theindividual.

In some embodiments, immune cells as described herein, once transferredto an individual, have a positive bystander effect on the endogenousimmune system by, i) producing proinflammatory cytokines, ii) recruitingadditional immune cells such as NK cells and APCs to the tumor site, andiii) inducing epitope spreading. Thus, in specific embodiments adoptivetransfer of the immune cells is useful to trigger a cascade of events invivo that amplifies the anti-tumor activity. In certain embodiments,even in the absence of use of the epigenetic modifier(s) and/ormitogenic agent, immune cells, e.g., dual targeted CAR-T cells, sufficeto reactivate a potent endogenous tumor-targeted immune response thatwill produce tumor elimination.

II. Immune Cells

Immune cells are utilized herein as the means by which a therapeuticprotein is delivered to a desired location in an individual in need oftherapy. The immune cells are modified such that they express at leastone therapeutic protein and, in specific embodiments, the therapeuticprotein allows targeting of the immune cells to a target that recognizesthe therapeutic protein. In specific embodiments, the therapeuticprotein is a receptor or an antibody on the immune cell and the targetis an antigen. In specific embodiments, the antigen is a cancer antigenand, if on a solid tumor cell, the antigen may be referred to as a tumorantigen.

In particular embodiments, methods and compositions for immunotherapyare provided, wherein the immunotherapy encompasses modified immunecells. The immune cells are modified to express one or more therapeuticproteins, the expression of which is in need of being maintained at alevel sufficient to render the cell effective for immunotherapy. Theimmune cells may be of any kind, although in specific embodiments theyare T cells. The immune cells are exposed, such as directly, with anagent that upregulates expression of the therapeutic protein(s). Theimmune cells may be contacted with an agent prior to delivery to anindividual in need thereof and/or the immune cells may be contacted withan agent subsequent to delivery to an individual in need thereof. Thecells may be contacted directly with the agent(s) in vitro or ex vivo,and/or the cells may be contacted in vivo with the agent upon systemicand/or localized delivery of the agent to an individual prior to,during, and/or after delivery of the cells to the individual.

In specific embodiments, the immune cells are T cells (e.g., CD4+ Tcells, CD8+ T cells, CD4+CD8+ T cells and/or Treg cells), or are NKcells or dendritic cells. As used herein, the term “immune cell”includes the primary subject cell and its progeny. It is understood thatprogeny are homogeneous but that progeny may not all be identical due todeliberate or inadvertent mutations. In the context of expressing aheterologous nucleic acid sequence, the immune cell is a cell that iscapable of replicating a vector and/or expressing a heterologous geneencoded by a vector. In particular embodiments, an immune cell isengineered to express an exogenous nucleic acid encoding a therapeuticprotein by transducing the cell with a viral vector comprising theexogenous nucleic acid. In certain embodiments, the viral vector is arretoviral vector. In one embodiment, the retroviral vector is alentiviral vector. An immune cell may be engineered to express anexogenous nucleic acid, e.g, a nucleic acid contained in a vector, e.g.,an expression vector. As used herein, “engineered” or “recombinant” cellrefers to a cell into which an exogenous nucleic acid sequence, such as,for example, a vector, has been introduced. Therefore, recombinant cellsare distinguishable from naturally occurring cells that do not contain arecombinantly introduced nucleic acid.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which the abovedescribed host cells can maintain and permit replication of at least onevector. Also understood and known are techniques and conditions thatwould allow large-scale production of vectors, as well as production ofthe nucleic acids encoded by vectors and their cognate polypeptides,proteins, or peptides.

The immune cells can be autologous cells, syngeneic cells, allogeniccells and even in some cases, xenogeneic cells.

In some embodiments, the cells harbor more than one therapeutic protein,and at least in certain aspects the exposure of the cells (or exposureto the individual receiving the cells) to an epigenetic modifier ormitogenic agent results in upregulation of expression of the two or moretherapeutic proteins.

In particular embodiments, the immune cells are genetically engineeredto express a therapeutic protein, e.g., a CAR, an engineered αβTCR,and/or antigen-specific receptor.

III. Immune Cells Comprising CAR(s)

In particular aspects, the immune cells are T cells that express achimeric antigen receptor (CAR). The use of CAR-modified T cells as atherapy for both hematologic malignancies and solid tumors is becomingmore widespread. However, the infusion of a T cell product targeting asingle tumor associated antigen (TAA) or tumor-specific antigen (TAA)may lead to target antigen modulation under this selective pressure, ormay select for tumor cell expressing low levels of the TAA or TSA, withsubsequent tumor immune escape. Tumor escape by the same mechanism mayoccur even when two TAAs or TSAs are targeted. Surprisingly, it has beenfound that the magnitude of tumor destruction depended not only on thepresence of the target antigen but also the intensity of expression.Expression of such TAAs or TSAs may be increased by administeringepigenetic modulators that upregulate target expression and enhanceCAR-T cell potency, in particular embodiments.

A chimeric antigen receptor (CAR) is an artificially constructed hybridprotein or polypeptide typically containing an extracellular antigenbinding domain, a transmembrane domain, and an intracellular signalingdomain. Characteristics of CARs include their ability to redirect T-cellspecificity and reactivity toward a selected target in anon-MHC-restricted manner, exploiting the antigen-binding properties ofmonoclonal antibodies. The non-MHC-restricted antigen recognition givesT cells expressing CARs the ability to recognize antigen independent ofantigen processing, thus bypassing a major mechanism of tumor escape.Moreover, when expressed in T-cells, CARs advantageously do not dimerizewith endogenous T cell receptor (TCR) alpha and beta chains.

The phrases “have antigen specificity” and “elicit antigen-specificresponse” as used herein means that the CAR can specifically bind to andimmunologically recognize an antigen, such that binding of the CAR tothe antigen elicits an immune response.

The extracellular antigen binding domain may be any protein or portionthereof that binds to a target protein, e.g., a receptor orligand-binding portion thereof; a ligand of a receptor (e.g., acytokine); or an antibody or antigen-binding portion of an antibody,e.g., an Fc domain of a single-chain antibody (scFv).

In particular embodiments, a CAR comprises a transmembrane domainselected from the group consisting of: a CD4 transmembrane domain, a CD8transmembrane domain, and a CD28 transmembrane domain.

The intracellular signaling domain, in certain embodiments, comprises aprimary signaling domain, e.g., a T cell receptor zeta chain or primarysignaling domain therefrom. In particular embodiments, the intracellularsignaling domain further comprises one or more co-stimulatory domains.Illustrative examples of co-stimulatory domains suitable for use in theCARs contemplated herein include, but are not limited to: e.g. CD27,CD28, CD137 (4-1BB), OX-40, or a combination of two, three, or all ofthe foregoing.

In specific embodiments, the CAR comprises an antibody for the tumorantigen, part or all of a cytoplasmic signaling domain, and/or part orall of one or more co-stimulatory molecules, for example endodomains ofco-stimulatory molecules. In specific embodiments, the antibody is asingle-chain variable fragment (scFv). In certain aspects the antibodyis directed at target antigens on the cell surface of cancer cells, forexample. In certain embodiments, a cytoplasmic signaling domain, such asthose derived from the T cell receptor zeta-chain, is employed as atleast part of the chimeric receptor in order to produce stimulatorysignals for T lymphocyte proliferation and effector function followingengagement of the chimeric receptor with the target antigen.Illustrative examples include, but are not limited to, endodomains fromco-stimulatory molecules such as CD27, CD28, 4-1BB, ICOS (CD278) andOX40, or combinations of two, three, four, or all of the foregoing. Inparticular embodiments, co-stimulatory molecules are employed to enhancethe activation, proliferation, and cytotoxicity of T cells produced bythe CAR after antigen engagement. In specific embodiments, theco-stimulatory molecules are CD28, OX40, and 4-1BB.

In one embodiment, the CAR comprises an extracellular hinge domain,transmembrane domain, and optionally, an intracellular hinge domaincomprising CD8 sequences and an intracellular T cell receptor signalingdomain comprising CD28, 4-1BB, and CD3ζ. CD28 is a T cell markerimportant in T cell co-stimulation. CD8 is also a T cell marker. 4-1BBtransmits a potent costimulatory signal to T cells, promotingdifferentiation and enhancing long-term survival of T lymphocytes. CD3 ζassociates with TCRs to produce a signal and contains immunoreceptortyrosine-based activation motifs (ITAMs). In one embodiment, a CARcomprises an extracellular hinge domain, transmembrane domain, andoptional intracellular hinge domain.

The CAR may be first generation, second generation, or third generation(CAR in which signaling is provided by CD3ζ together with co-stimulationprovided by CD28 and a tumor necrosis factor receptor (TNFr), such as4-1BB or OX40), for example. The CAR may be specific for PSCA, HER2,CD19, CD20, CD22, Kappa or light chain, Lambda, CD30, CD33, CD123, CD38,ROR1, ErbB2, ErbB3/4, EGFR, EGFRvIII, EphA2, FAP, carcinoembryonicantigen, EGP2, EGP40, mesothelin, TAG72, PSMA, NKG2D ligands, B7-H6,IL-13 receptor α2, IL-11 receptor α, MUC1, MUC16, CA9, CE7, CEA, GD2,GD3, HMW-MAA, CD171, Lewis Y, G250/CAIX, HLA-AI MAGE A1, HLA-A2NY-ESO-1, PSC1, folate receptor-α, CD44v7/8, 8H9, NCAM, VEGF receptors,5T4, Fetal AchR, NKG2D ligands, CD44v6, TEM1, TEM8, sp17,viral-associated antigens expressed by the tumor, or othertumor-associated antigens that are identified through genomic analysisand or differential expression studies of tumors.

In particular embodiments, the CAR is encoded by an expression vector.The vector may be bicistronic, in particular embodiments. In someembodiments, more than one CAR is expressed by the immune cell. Inparticular embodiments where more than one CAR is to be expressed by theimmune cell, the two or more CAR expression constructs may or may not beon the same vector. When present on the same vector, the first CARcoding sequence may be configured 5′ or 3′ to the second CAR codingsequence. The expression of the first CAR and second or subsequent CARreceptor may be under the direction of the same or different regulatorysequences.

In particular cases, the immune cell comprises the therapeutic proteinas a membrane-bound protein. In certain embodiments, the protein issecretable from the immune cell. In particular embodiments, thetherapeutic protein is a receptor for a cancer antigen; the cancerantigen (which may be on a solid tumor or not) may be present on thesurface of a cancer cell. In specific embodiments, the receptor is achimeric antigen receptor (CAR). In particular cases, the immune cell isa T cell comprising one, two, three, or more CARs.

In some embodiments wherein the immune cell comprises at least one CAR,the CAR may be directed to any type of cancer antigen. In particularembodiments, the immune cell comprises one CAR directed to one cancerantigen, and another CAR in the same cell directed to another cancerantigen.

In certain embodiments, an individual is provided a therapeuticallyeffective amount of a plurality of immune cells expressing one or moretherapeutic proteins. In some embodiments, the individual issubsequently provided a therapeutically effective amount of a pluralityof immune cells expressing one or more other therapeutic proteinsdifferent from that (or those) initially provided to the individual.

In some situations one may wish to be able to kill the modified immunecellse.g. In particular embodiments, the expression of certain geneproducts kills the immune cells under controlled conditions, such asinducible suicide genes. Illustrative examples of inducible suicidegenes include, but are not limited to: caspase-9 Herpes Simplex Virus(HSV) thymidine kinase (TK) gene, cytosine daminase, purine nucleosidephosphorylase, and nitroreductase.

IV. Agents that Upregulate Expression of a Therapeutic Protein

In various embodiments, an agent that upregulates expression of thetherapeutic protein in an immune cell is provided. In certainembodiments, the upregulation is detectable and greater than thatexpression in the cell in the absence of exposure to the agent. Inspecific embodiments, the agent(s) upregulates expression of thetherapeutic protein at least two-fold, three-fold, four-fold, five-fold,ten-fold, twenty-fold, twenty five-fold, thirty-fold, thirty five-fold,forty-fold, forty five-fold, fifty-fold, one hundred-fold, twohundred-fold, five hundred-fold, one thousand-fold, or more.

In one embodiment, the agent that upregulates expression may be of anykind. In specific embodiments the agent is one or both of an epigeneticmodifier, including, but not limited to, an HDAC inhibitor and/or a DNMTinhibitor, and a mitogen.

A. Epigenetic Modifiers

In some embodiments, one or more epigenetic modifiers are utilized inmethods and compositions to upregulate expression of one or moretherapeutic proteins in an immune cell. The epigenetic modifiers may beof any kind so long as they are capable of upregulating expression of atleast one therapeutic protein in an immune cell. One or more types ofepigenetic modifiers may be used in the same method or composition. Whenmore than one epigenetic modifier is employed, they may be delivered tocells or to an individual at the same time, at different times, in thesame formulation, or in different formulations. In some cases, theepigenetic modifier is a histone deacetylase (HDAC) inhibitor. In somecases, the epigenetic modifier is DNA methyltransferase (DNMT)inhibitor. In certain cases, a combination of HDAC inhibitor and DNMTinhibitor is employed. The epigenetic modifier may be an EZH2antagonist, such as DZnep or 3-deazaneplanocin A, for example.

In some embodiments, the epigenetic modifier(s) comprises one or morehistone deacetylase (HDAC) inhibitors. The HDAC inhibitors include, butare not limited to small chain fatty acids, hyroxamic acids, cyclicpeptides, or benzamides. Further illustrative examples of HDACinhibitors include, but are not limited to, trichostatin A, sodiumphenylbutyrate, Buphenyl, Ammonaps, Valproic acid, Depakote, romidepsin(ISTODAX®), Vorinostat, Zolinza, panobinostat, belinostat, entinostat,JNJ-26481585 (Johnson & Johnson; Langhorne, Pa.), and/or MGCD-0103(MethylGene; Montreal, Canada).

In some embodiments, the epigenetic modifier(s) comprises one or moreDNMT inhibitors. The DNMT inhibitors include, but are not limited tonucleoside analogs, quinolone, or active site inhibitors. Furtherillustrative examples of DNMT inhibitors include but are not limited to5-azacitidine (such as VIDAZA®), decitabine (e.g., DACOGEN®),zebularine, SGI-110 or SGI-1036 (SuperGen; Dublin, Calif.), RG108,caffeic acid purum, chlorogenic acid, epigallocatechin galiate,procainamide hydrochloride, a procainamide derivative,5-azadeoxycytidine, 5′-aza-2′-deoxycytidine or MG98.

B. Mitogens

In some embodiments, one or more mitogens are used in methods orcompositions. The mitogen(s) may be provided to an individual in needthereof or to immune cells that are to be delivered to an individual inneed thereof. The mitogen may be used as the only type of agent, or themitogen may be used in combination with another agent, including a HDACinhibitor and/or DNMT inhibitor, for example. Illustrative examples ofmitogens include but are not limited to concanavalin A,phytohaemagglutinin, lipopolysaccharide, and pokeweed mitogen.

V. Delivery of the Agent(s) to Cells and/or Individuals

In particular embodiments, in vivo or in vitro or ex vivo methods areprovided and in some embodiments part of the method may be in vitro orex vivo, followed by an in vivo step, for example.

In particular in vitro embodiments, a plurality of immune cells may beobtained. The cells may be obtained from an individual and manipulatedand ultimately delivered back into the same individual. The cells may beobtained from an individual, manipulated, and ultimately delivered intoanother individual. In some cases, the immune cells are obtained from arepository or commercially, for example.

Prior to exposure of the cells to an agent, the immune cells may bemanipulated in one or more of a variety of ways. In specificembodiments, a nucleic acid is introduced into the cells, such as bystandard means. The nucleic acid may be at least one vector with atleast one expression construct that encodes at least one therapeuticprotein, for example. In particular embodiments, the therapeutic proteinis a receptor, cytokine, ligand trap, or antibody (including monomericor multimeric). In specific embodiments, the therapeutic protein is achimeric antigen receptor (CAR) or cytokine receptor.

In some embodiments, the cells are manipulated such that they encompassmore than one therapeutic protein, and there may be mechanisms tomonitor retention of the two or more therapeutic proteins in thecell(s), such as labels and/or selectable markers.

In particular embodiments, the cells of the in vitro or ex vivo methodare expanded, such as by routine methods in the art Exposure of thecells to an epigenetic modifier agent or mitogenic agent may occur byany suitable regimen so long as the cells receive a sufficient amount ofthe agent and for a sufficient time to upregulate expression of thetherapeutic protein. In some embodiments, the agent is delivered to theimmune cells more than once prior to delivery of the cells to theindividual. In certain embodiments, more than one agent is provided tothe immune cells at the same or different times prior to the delivery ofthe cells to the individual, and the agent may be of the same ordifferent types, such as one HDAC inhibitor and one DNMT inhibitor, forexample. When an individual is pre-treated with an epigenetic modifieragent or mitogenic agent prior to the delivery of the cells to theindividual, that agent or agents may or may not be the same agent oragents provided to the cells prior to delivery to the individual. Insome cases, the cells are not exposed to an epigenetic modifier agent ormitogenic agent prior to the delivery of the cells to the individual.

In particular embodiments, an individual in need of treatment with theimmune cells/agent are exposed to the agent prior to receipt of thecells, although in specific embodiments, the individual may be exposedto the agent following receipt of the cells in addition to or as analternative to exposure to the agent prior to receipt of the cells. Inparticular embodiment, the individual is provided with pre-treatment ofthe agent in multiple doses. The agent may be delivered to theindividual by any suitable means, although in specific embodiments thedelivery is oral, subcutaneous, intravenous, intramuscular,intraperitoneal, buccal, and so forth. When more than one agent isdelivered to the individual, the agents may be delivered by separatedelivery means, although in particular embodiments, the agents aredelivered by the same route and may or may not be in the sameformulation.

In some embodiments, the individual receives the immune cells multipletimes, and the separate deliveries may be separated by a space of timeof minutes, days, weeks, months, or years. In these cases, the separatecourses of delivery of the cells may encompass immune cells having thesame or different therapeutic proteins. The individual may in subsequentrounds be exposed to treatment of the same or different agent than theagent that the individual was treated with in the initial round. Anyround of exposure of the cells to the individual may encompasspre-treatment and/or post-treatment with the agent.

VI. Pharmaceutical Compositions

With respect to pharmaceutical compositions comprising the agents and/orthe cells, the pharmaceutically acceptable carrier can be any of thoseconventionally used and is limited only by chemico-physicalconsiderations, such as solubility and lack of reactivity with theactive agent(s), and by the route of administration. Thepharmaceutically acceptable carriers described herein, for example,vehicles, adjuvants, excipients, and diluents, are well-known to thoseskilled in the art and are readily available to the public. It ispreferred that the pharmaceutically acceptable carrier be one which ischemically inert to the active agent(s) and one which has no detrimentalside effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particularmaterial, as well as by the particular method used to administer theinventive material. Accordingly, there are a variety of suitableformulations of the pharmaceutical composition of the invention.Preservatives may be used. Suitable preservatives may include, forexample, methylparaben, propylparaben, sodium benzoate, and benzalkoniumchloride. A mixture of two or more preservatives optionally may be used.The preservative or mixtures thereof are typically present in an amountof about 0.0001% to about 2% by weight of the total composition.

Suitable buffering agents may include, for example, citric acid, sodiumcitrate, phosphoric acid, potassium phosphate, and various other acidsand salts. A mixture of two or more buffering agents optionally may beused. The buffering agent or mixtures thereof are typically present inan amount of about 0.001% to about 4% by weight of the totalcomposition.

Methods for preparing administrable (e.g., parenterally administrable)compositions are known or apparent to those skilled in the art and aredescribed in more detail in the Physicians Desk Reference, 62nd edition.Oradell, N.J.: Medical Economics Co., 2008; Goodman & Gilman's ThePharmacological Basis of Therapeutics, Eleventh Edition. McGraw-Hill,2005; Remington: The Science and Practice of Pharmacy, 21^(st) Edition.Baltimore, Md.: Lippincott Williams & Wilkins, 2005; and The MerckIndex, Fourteenth Edition. Whitehouse Station, N.J.: Merck ResearchLaboratories, 2006; each of which is hereby incorporated by reference inrelevant parts.

The following formulations for oral, aerosol, parenteral (e.g.,subcutaneous, intravenous, intraarterial, intramuscular, intradermal,interperitoneal, and intrathecal), and topical administration are merelyexemplary and are in no way limiting. More than one route can be used toadminister the inventive CAR materials, and in certain instances, aparticular route can provide a more immediate and more effectiveresponse than another route.

Formulations suitable for oral administration include liquid solutionsoptionally comprising diluents, such as water, saline, or orange juice;(b) capsules, sachets, tablets, lozenges, and troches, each containing apredetermined amount of the active ingredient, as solids or granules;(c) powders; (d) suspensions in an appropriate liquid; and (e) suitableemulsions. Liquid formulations may include diluents, such as water andalcohols, for example, ethanol, benzyl alcohol, and the polyethylenealcohols, either with or without the addition of a pharmaceuticallyacceptable surfactant. Capsule forms can be of the ordinary hard orsoftshelled gelatin type containing, for example, surfactants,lubricants, and inert fillers, such as lactose, sucrose, calciumphosphate, and corn starch. Tablet forms can include one or more oflactose, sucrose, mannitol, corn starch, potato starch, alginic acid,microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicondioxide, croscarmellose sodium, talc, magnesium stearate, calciumstearate, zinc stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, disintegrating agents, moistening agents,preservatives, flavoring agents, and other pharmacologically compatibleexcipients. Lozenge forms can comprise flavoring compounds, usuallysucrose and acacia or tragacanth, as well as pastilles in an inert base,such as gelatin and glycerin, or sucrose and acacia, emulsions, gels,and the like containing, in addition to, such excipients as are known inthe art.

Formulations suitable for parenteral administration include aqueous andnonaqueous isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and nonaqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The inventive. CAR material can be administered in a physiologicallyacceptable diluent in a pharmaceutical carrier, such as a sterile liquidor mixture of liquids, including a pharmaceutically acceptable cellculture medium, water, saline, aqueous dextrose and related sugarsolutions, an alcohol, such as ethanol or hexadecyl alcohol, a glycol,such as propylene glycol or polyethylene glycol, dimethylsulfoxide,glycerol, ketals such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers,poly(ethyleneglycol) 400, oils, fatty acids, fatty acid esters orglycerides, or acetylated fatty acid glycerides with or without theaddition of a pharmaceutically acceptable surfactant, such as a soap ora detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-β-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations will typically contain, for example, fromabout 0.5% to about 25% by weight of the active agents or cells insolution. Preservatives and buffers may be used. In order to minimize oreliminate irritation at the site of injection, such compositions maycontain one or more nonionic surfactants having, for example, ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations will typically range, forexample, from about 5% to about 15% by weight. Suitable surfactantsinclude polyethylene glycol sorbitan fatty acid esters, such as sorbitanmonooleate and the high molecular weight adducts of ethylene oxide witha hydrophobic base, formed by the condensation of propylene oxide withpropylene glycol. The parenteral formulations can be presented inunit-dose or multi-dose sealed containers, such as ampoules and vials,and can be stored in a freeze-dried (lyophilized) condition requiringonly the addition of the sterile liquid excipient, for example, water,for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions can be prepared from sterile powders,granules, and tablets of the kind previously described.

Injectable formulations are in accordance with an embodiment of theinvention. The requirements for effective pharmaceutical carriers forinjectable compositions are well-known to those of ordinary skill in theart (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. LippincottCompany, Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250(1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages622-630 (1986)).

Topical formulations, including those that are useful for transdermaldrug release, are well known to those of skill in the art and aresuitable in the context of embodiments of the invention for applicationto skin. The compositions contemplated herein can be made into aerosolformulations to be administered via inhalation. These aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like. They alsomay be formulated as pharmaceuticals for non-pressured preparations,such as in a nebulizer or an atomizer. Such spray formulations also maybe used to spray mucosa.

An “effective amount” or “an amount effective to treat” refers to a dosethat is adequate to prevent or treat cancer in an individual. Amountseffective for a therapeutic or prophylactic use will depend on, forexample, the stage and severity of the disease or disorder beingtreated, the age, weight, and general state of health of the patient,and the judgment of the prescribing physician. The size of the dose willalso be determined by the active selected, method of administration,timing and frequency of administration, the existence, nature, andextent of any adverse side-effects that might accompany theadministration of a particular active, and the desired physiologicaleffect. It will be appreciated by one of skill in the art that variousdiseases or disorders could require prolonged treatment involvingmultiple administrations, perhaps using the agents and/or cellscontemplated herein, in each or various rounds of administration. By wayof example and not intending to limit the invention, the dose of thecompositions can be about 0.001 to about 1000 mg/kg body weight of thesubject being treated/day, from about 0.01 to about 10 mg/kg bodyweight/day, about 0.01 mg to about 1 mg/kg body weight/day.

It can generally be stated that a pharmaceutical composition comprisingthe T cells described herein may be administered at a dosage of 10² to10¹⁰ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg body weight,including all integer values within those ranges. The number of cellswill depend upon the ultimate use for which the composition is intendedas will the type of cells included therein. For uses provided herein,the cells are generally in a volume of a liter or less, can be 500 mLsor less, even 250 mLs or 100 mLs or less. Hence the density of thedesired cells is typically greater than 10⁶ cells/ml and generally isgreater than 10⁷ cells/ml, generally 10⁸ cells/ml or greater. Theclinically relevant number of immune cells can be apportioned intomultiple infusions that cumulatively equal or exceed 10⁵, 10⁶, 10⁷, 10⁸,10⁹, 10¹⁰, 10¹¹, or 10¹² cells. In some aspects of the presentinvention, particularly since all the infused cells will be redirectedto a particular target antigen, lower numbers of cells, in the range of10⁶/kilogram (10⁶-10¹¹ per patient) may be administered. CAR expressingcell compositions may be administered multiple times at dosages withinthese ranges.

For purposes of the invention, the amount or dose of compositionscontemplated herein administered should be sufficient to effect atherapeutic or prophylactic response in the subject or animal over areasonable time frame. For example, the dose of a composition should besufficient to bind to antigen, or detect, treat or prevent disease in aperiod of from about 2 hours or longer, e.g., about 12 to about 24 ormore hours, from the time of administration. In certain embodiments, thetime period could be even longer. The dose will be determined by theefficacy of the particular composition and the condition of the animal(e.g., human), as well as the body weight of the animal (e.g., human) tobe treated.

Delivery systems useful in the context of embodiments contemplatedherein may include time-released, delayed release, and sustained releasedelivery systems such that the delivery of the inventive compositionoccurs prior to, and with sufficient time to cause, sensitization of thesite to be treated. The inventive composition can be used in conjunctionwith other therapeutic agents or therapies. Such systems can avoidrepeated administrations of the composition, thereby increasingconvenience to the subject and the physician, and may be particularlysuitable for certain composition embodiments of the invention.

Many types of release delivery systems are available and known to thoseof ordinary skill in the art. They include polymer base systems such aspoly(lactide-glycolide), copolyoxalates, polycaprolactones,polyesteramides, polyorthoesters, polyhydroxybutyric acid, andpolyanhydrides. Microcapsules of the foregoing polymers containing drugsare described in, for example, U.S. Pat. No. 5,075,109. Delivery systemsalso include non-polymer systems that are lipids including sterols suchas cholesterol, cholesterol esters, and fatty acids or neutral fats suchas mono-di- and tri-glycerides; hydrogel release systems; sylasticsystems; peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which the active composition is contained in a form within amatrix such as those described in U.S. Pat. Nos. 4,452,775, 4,667,014,4,748,034, and 5,239,660 and (b) diffusional systems in which an activecomponent permeates at a controlled rate from a polymer such asdescribed in U.S. Pat. Nos. 3,832,253 and 3,854,480. In addition,pump-based hardware delivery systems can be used, some of which areadapted for implantation.

VII. Combination Treatments

In certain embodiments, one or more medical treatments may be providedto an individual in addition to the immune cells and/or epigeneticmodifying or mitogenic agent that themselves comprises a therapeuticagent. The one or more other medical treatments may be suitable for anykind of medical condition, but in particular embodiments the medicalcondition is cancer.

In specific embodiments, the combination therapy comprises one or moreanti-cancer agents. An “anti-cancer” agent is capable of negativelyaffecting cancer in a subject, for example, by killing cancer cells,inducing apoptosis in cancer cells, reducing the growth rate of cancercells, reducing the incidence or number of metastases, reducing tumorsize, inhibiting tumor growth, reducing the blood supply to a tumor orcancer cells, promoting an immune response against cancer cells or atumor, preventing or inhibiting the progression of cancer, or increasingthe lifespan of a subject with cancer. More generally, these othercompositions would be provided in a combined amount effective to kill orinhibit proliferation of the cell. This process may involve contactingthe cells with the nanoparticle and the agent(s) or multiple factor(s)at the same time. This may be achieved by contacting the cell with asingle composition or pharmacological formulation that includes bothagents, or by contacting the cell with two distinct compositions orformulations, at the same time, wherein one composition includes thenanoparticles and the other includes the second agent(s).

In the context of the present invention, it is contemplated that thepresent therapy could be used similarly in conjunction withchemotherapeutic, radiotherapeutic, or immunotherapeutic intervention,for example. Alternatively, the present therapy may precede or followthe other treatment by intervals ranging from minutes to weeks. In someembodiments where the other therapy and present therapy are appliedseparately to the cell or individual, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the agent and present therapy would still be able toexert an advantageously combined effect on the cell. In such instances,it is contemplated that one may contact the cell with both modalitieswithin about 12-24 h of each other and, more preferably, within about6-12 h of each other. In some situations, it may be desirable to extendthe time period for treatment significantly, however, where several d(2, 3, 4, 5, 6 or 7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapsebetween the respective administrations.

Various combinations may be employed, such as wherein nanoparticletherapy is “A” and the secondary agent, such as radio- or chemotherapy,is “B”:

A/B/A  B/A/B  B/B/A  A/A/B  A/B/B  B/A/A  A/B/B/B  B/A/B/BB/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/AB/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Administration of the therapeutic nanoparticles of the present inventionto a patient will follow general protocols for the administration ofchemotherapeutics, in some cases. It is expected that the treatmentcycles would be repeated as necessary. It also is contemplated thatvarious standard therapies, as well as surgical intervention, may beapplied in combination with the present hyperproliferative therapy.

A. Chemotherapy

Cancer therapies also include a variety of combination therapies withboth chemical and radiation based treatments. Combination chemotherapiesinclude, for example, cisplatin (CDDP), carboplatin, procarbazine,mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan,chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin,doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16),tamoxifen, raloxifene, estrogen receptor binding agents, taxol,gemcitabien, navelbine, farnesyl-protein tansferase inhibitors,transplatinum, 5-fluorouracil, vincristine, vinblastin and methotrexate,or any analog or derivative variant of the foregoing.

B. Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageon DNA, on the precursors of DNA, on the replication and repair of DNA,and on the assembly and maintenance of chromosomes. Dosage ranges forX-rays range from daily doses of 50 to 200 roentgens for prolongedperiods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.Dosage ranges for radioisotopes vary widely, and depend on the half-lifeof the isotope, the strength and type of radiation emitted, and theuptake by the neoplastic cells.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

C. Immunotherapy

An immunotherapy other than that of the present disclosure may beemployed in addition in particular embodiments. Immunotherapeutics,generally, rely on the use of immune effector cells and molecules totarget and destroy cancer cells, and immunotherapies other than thepresent invention may be employed in addition to the presentembodiments. The alternative therapy may or may not be comprised on theimmune cells contemplated herein.

The immune effector may be, for example, an antibody specific for somemarker on the surface of a tumor cell. The antibody alone may serve asan effector of therapy or it may recruit other cells to actually effectcell killing. The antibody also may be conjugated to a drug or toxin(chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussistoxin, etc.) and serve merely as a targeting agent. Alternatively, theeffector may be a lymphocyte carrying a surface molecule that interacts,either directly or indirectly, with a tumor cell target. Variouseffector cells include cytotoxic T cells and NK cells.

Immunotherapy, thus, could be used as a two-pronged approach for thecombined therapy. The general approach for combined therapy is discussedbelow. Generally, the tumor cell must bear some marker that is amenableto targeting, i.e., is not present on the majority of other cells. Manytumor markers exist and any of these may be suitable for targeting inthe context of the present invention. Common tumor markers includecarcinoembryonic antigen, prostate specific antigen, urinary tumorassociated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG,Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, lamininreceptor, erb B and p155.

D. Gene Therapy

In yet another embodiment, the secondary treatment may be a gene therapyin which a therapeutic polynucleotide is administered before, after, orat the same time as the present therapy. The therapeutic polynucleotidemay encode all of part of a therapeutic polypeptide or thepolynucleotide may be therapeutic itself (such as miRNA, siRNA, shRNA).Delivery of a vector encoding either a full length or truncatedtherapeutic polypeptide or a therapeutic polynucleotide in conjuctionwith therapy of the present disclosure will have a combinedanti-hyperproliferative effect on target tissues.

E. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs'surgery). It is further contemplated that the present invention may beused in conjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

F. Other agents

It is contemplated that other agents may be used in combination with thepresent invention to improve the therapeutic efficacy of treatment.These additional agents include immunomodulatory agents, agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adhesion, oragents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers. Immunomodulatory agents include tumor necrosisfactor; interferon alpha, beta, and gamma; IL-2 and other cytokines;F42K and other cytokine analogs; or MIP-1, MIP-1beta, MCP-1, RANTES, andother chemokines. It is further contemplated that the upregulation ofcell surface receptors or their ligands such as Fas/Fas ligand, DR4 orDRS/TRAIL would potentiate the apoptotic inducing abililties of thepresent invention by establishment of an autocrine or paracrine effecton hyperproliferative cells. Increases intercellular signaling byelevating the number of GAP junctions would increase theanti-hyperproliferative effects on the neighboring hyperproliferativecell population. In other embodiments, cytostatic or differentiationagents can be used in combination with the present invention to improvethe anti-hyerproliferative efficacy of the treatments. Inhibitors ofcell adehesion are contemplated to improve the efficacy of the presentinvention. Examples of cell adhesion inhibitors are focal adhesionkinase (FAKs) inhibitors and Lovastatin. It is further contemplated thatother agents that increase the sensitivity of a hyperproliferative cellto apoptosis, such as the antibody c225, could be used in combinationwith the present invention to improve the treatment efficacy.

Hormonal therapy may also be used in conjunction with the presentinvention or in combination with any other cancer therapy previouslydescribed. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen. This treatment is often used in combinationwith at least one other cancer therapy as a treatment option or toreduce the risk of metastases.

VIII. Kits

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, there may be an epigenetic modifier and/ormitogenic agent and/or immune cells and/or an apparatus for extractingimmune's cells that are autologous or allogeneic compared to anindividual. One or more therapeutic or other agents may be comprised ina kit, such as another type of therapy, including a drug(s), such as acancer drug. In some embodiments, reagents for expansion of immune cellsmay be included. Other compositions may include standard buffers, salts,and the like. The kit will comprise its components in suitable containermeans. Such components may be suitably aliquoted. The components of thekits may be packaged either in aqueous media or in lyophilized form. Thecontainer means of the kits will generally include at least one vial,test tube, flask, bottle, syringe or other container means, into which acomponent may be placed, and preferably, suitably aliquoted. Where thereare more than one component in the kit, the kit also will generallycontain a second, third or other additional container into which theadditional components may be separately placed. However, variouscombinations of components may be comprised in a container. The kits ofthe present invention also will typically include a means for containingthe component containers in close confinement for commercial sale. Suchcontainers may include injection or blow-molded plastic containers intowhich the desired vials are retained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution may be an aqueous solution, with asterile aqueous solution being particularly preferred. The compositionsmay also be formulated into a syringeable composition. In which case,the container means may itself be a syringe, pipette, and/or other suchlike apparatus, from which the formulation may be applied to an infectedarea of the body, injected into an animal, and/or even applied to and/ormixed with the other components of the kit.

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans.

Irrespective of the number and/or type of containers, the kits of theinvention may also comprise, and/or be packaged with, an instrument forassisting with the injection/administration and/or placement of theultimate composition within the body of an animal. Such an instrumentmay be a syringe, pipette, forceps, and/or any such medically approveddelivery vehicle.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Methods and Materials

Exemplary materials and methods are provided herein.

Donor and Cell Lines

The pancreatic cancer cell line, CAPAN1, which naturally express PSCAand MUC1, and human embryonic kidney cell line, 293T, were obtained fromthe American Type Culture Collection (ATCC; Rockville, Md.). Cells weremaintained in complete IMDM media (IMDM; Gibco by Life TechnologiesCorporation, Grand Island, N.Y.), 10% FBS (Hyclone Laboratories, Inc.,Logan, Utah) and 2 mM L-glutaMAX (Gibco by Life TechnologiesCorporation, Grand Island, N.Y.) and a humidified atmosphere containing5% carbon dioxide (CO₂) at 37° C. Peripheral blood mononuclear cells(PBMCs) from healthy volunteers were obtained with informed consent onprotocols approved by the Baylor College of Medicine InstitutionalReview Board.

OKT3/CD28 Blast Generation

PBMCs obtained from healthy donors were activated with OKT3 (1 mg/ml)(Ortho Biotech, Inc., Bridgewater, N.J.) and CD28 antibodies (1 mg/ml)(Becton Dickinson & Co., Mountain View, Calif.) and plated in a nontissue culture-treated 24-well plate at 1×10⁶ PBMCs/2 ml in completemedia (RPMI 1640; Hyclone Laboratories, Inc., Logan, Utah) containing45% Clicks medium (Irvine Scientific, Inc., Santa Ana, Calif.), 10% FBS(Hyclone Laboratories, Inc., Logan, Utah) and 2 mM L-glutaMAX (Gibco byLife Technologies Corporation, Grand Island, N.Y.) and subsequentlysplit and fed with fresh media plus IL2 (50 U/ml).

Generation of Retroviral Constructs and Retroviral Transduction

A codon-optimized single chain variable fragment (scFV) of MUC1 and ahumanized, codon-optimized scFV of PSCA were synthesized (DNA 2.0, MenloPark, Calif.) based on published sequences. The scFV fragments werecloned in-frame with the human IgG1-CH2CH3 domain and with the ζ-chainof the TCR/CD3 complex in the SFG retroviral backbone, to make firstgeneration CAR-PSCA and CAR-MUC1 retroviral constructs. In order todistinguish CAR-modified T cells ΔCD19 (T cells modified to express abeta cell marker, for ease of identification) was incorporated into theCAR-MUC1 retroviral vector using an IRES element. To generate 2^(nd) and3^(rd) generation CAR-PSCA constructs the CD28 endodomain or CD28 and41BB costimulatory endodomains were added to the first generation CARbetween the IgG1-CH2CH3 domain and the TCR/CD3ζ endodomain. Alsosynthesized (DNA 2.0, Menlo Park, Calif.) were the TAAs MUC1 and PSCA,based on published sequences. The fluorescent markers mOrange and greenfluorescence protein (GFP) were incorporated into the MUC1 antigen andthe PSCA antigen vectors, respectively, again using an IRES element.Retroviral supernatant was produced as previously described, filtered(using a 0.45-mm filter) and stored at −80° C.

T Cell Transduction

For T cell transduction, CAR-MUC1 or CAR-PSCA retroviral supernatant wasplated in a non-tissue culture-treated 24-well plate (1 ml/well), whichwas pre-coated with a recombinant fibronectin fragment (FN CH-296;Retronectin; Takara Shuzo Co. Ltd, Otsu, Japan). OKT3/CD28-activated Tcells (0.2×10⁶/ml in complete media with IL2 100 U/ml) were added to theplates (1 ml/well), and then transferred to a 37° C., 5% CO₂ incubator.CAR-T cell expansion was performed in a G-Rex 100M (Wilson WolfManufacturing; New Brighton, Minn.) with 1 L of complete mediasupplemented with IL2 (50 U/ml).

293T Transduction

For transduction, MUC1-mOrange or PSCA-GFP viral supernatant was platedin a retronectin pre-coated 24-well plate (1 ml/well). 0.2×10⁵/ml 293Tcells were added to the supernatant (1 ml/well), then the cells werespun at 1000 g for 30 min at room temperature (RT), and transferred to a37° C., 5% CO₂ incubator. Expression of MUC1-mOrange or PSCA-GFP wasmeasured 72 hrs post-transduction by flow cytometry and using afluorescence microscope to detect the fluorescent markers mOrange andGFP. Cells were maintained or expanded in complete IMDM media every 3-4days.

Cell Sorting

293T cells were sorted, based on mOrange and GFP expression, using aMoFlo flow cytometer (Cytomation, Fort Collins, Colo.). Sorted cellswere cultured in complete IMDM media supplemented with penicillin (100U/ml), streptomycin (100 μg/ml) and gentamicin (25 μg/ml) (Gibco by LifeTechnologies Corporation, Grand Island, N.Y.) for one week in a 6-wellplate, then further expanded in a T175 flask using complete IMDM media,which was replenished every 3-4 days.

Immunohistochemistry (IHC)

CAPAN1 cells were stained as described previously with either mouseanti-human MUC1 antibody or rabbit anti-human PSCA antibody (AbCam Inc.,Cambridge, Mass.) diluted 1:200 and 1:80, respectively, in PBS/1% bovineserum albumin (BSA) for 1 hr at RT and co-stained with anti-mousehorseradish peroxidase (HRP) or anti-rabbit HRP (AbCam Inc., Cambridge,Mass.).

Cytotoxicity

Chromium Release Assay

The cytotoxicity specificity of effector T cell populations was measuredin a standard 6 hr ⁵¹Cr release assay, using E:T ratios ranging from40:1 to 5:1, and using CAPAN1 and 293T cells as targets.

Co-Culture Experiment

CAPAN1, 293T, 293T-MUC1-mOrange or 293T-PSCA-GFP, were used as targets.Briefly, GFP/CAPAN1 cells were mixed with either OKT3/CD28 blasts orCAR-modified T cells at a 1:5 ratio in the presence of IL2 (50 U/ml) incomplete media. For our engineered tumor model, 293T-MUC1-mOrange and293T-PSCA-GFP (or control 293T cells alone) were mixed at 1:1 ratio thenOKT3/CD28 blasts or CAR-modified T cells were added to the mixture; 10:1(T cells:tumor cell), in the presence of IL2 (50 U/ml) in completemedia. After 72 hours all residual cells were collected, counted,stained and then analyzed by flow cytometry (Gallios; Beckman CoulterInc., Brea, Calif.).

Flow Cytometry

Immunophenotyping

T cells were analyzed 3-4 weeks after the generation of the culture bysurface-stained with monoclonal antibodies to: CD3, CD4, CD8, CD19,CD56, CD27, CD28, CD45RO, and CD62L (Becton Dickinson BD, FranklinLakes, N.J.). Cells were washed once with PBS supplemented with 2% FBS,pelleted, and antibodies added in saturating amounts (10 ul). To detectCAR-transduced cells, T cells were stained with a monoclonal antibodyFc-specific cyanine-Cy5-conjugated antibody (Jackson Immuno ResearchLaboratories, Inc., West Grove, Pa.), which recognizes the IgG1-CH2CH3component of the receptor. Cells were analyzed using a Gallios Flowcytometer and the data analyzed using Kaluza software (Beckman CoulterInc., Brea, Calif.).

MUC1 Antigen Staining

One million CAPAN1 cells were fixed with 80% methanol and washed with0.1% tween-PBS. 1 ug of anti-MUC1 antibody (Abcam Inc, Cambridge, Mass.)was added and incubated at RT for 30 mins. Then cultures were washed andincubated with 0.4 ug of a goat anti-mouse IgG APC antibody (BDPharmingen, San Jose, Calif.) for 20 mins at 4° C. in the dark. Cellswere then washed twice and analyzed.

In Vivo Study

One million CAPAN1 cells, which were engineered to express eGFP-Fireflyluciferase (eGFP-FFLuc), were inoculated intraperitoneally (IP) intoSCID mice. Bioluminescence images were recorded once a week using LuminaIVIS imaging system (Caliper Life Sciences Inc., Hopkinton, Mass.), andanalyzed by Living Image software. After engraftment, defined as anincrease in tumor signal in at least two consecutive bioluminescencemeasurements, mice were treated IP with CAR-modified T cells (30×10⁶cells/animal). All treated groups received IL-2 (4,000 U/animal) IPthree times per week and bioluminescence imaging was done once a week.

Decitabine Treatment

CAPAN1 cells were culture in a T175 flask using complete IMDM mediacontained 1 μM 5-Aza-2′-deoxycytidine—decitabine—(Sigma-aldrich Inc.,Saint Louis, Mo.) for 4 days, with fresh media+decitabine replenisheddaily. Subsequently, decitabine-treated CAPAN1 cells were rested for 2days in complete IMDM, and then co-cultured with CAR-MUC1 T cells.

Example 2 T Cells Engineered to Express a Car Targeting PSCA can KillAntigen-Expressing Targets

To target tumors expressing the TAA PSCA, a retroviral vector encoding ahumanized, codon-optimized CAR-directed against PSCA was generated. FIG.1 a shows the retroviral vector map and FIG. 1 b (left panel) shows CARexpression on T cells from a representative donor, and in summary forall 10 donors studied (FIG. 1 b, right panel). A mean of 89.9% (±9% SD)T cells expressed CAR-PSCA, and phenotype was unaffected by CARtransduction (FIG. 1 c) so that both non-transduced (NT) and CAR-PSCAtransgenic T cells were predominantly CD3+(95.2±5.7% and 95.2±3.5%),with a mixture of CD4+(19.2±12.0% and 12.8±6.3%) and CD8+(76.1±15.5% and82.2±10.5%) populations. CD56+ CD3− NK cells comprised 1.7±3.0% and2.7±2.2% of the NT and CAR-PSCA transduced populations. The sameproportion of CD3+ T cells in both NT and transduced populationsexpressed the central memory markers CD62L, CD27 and CD45RO. FIG. 1 ddemonstrates that CAR-modified T cells were able to kill PSCA+pancreatic cancer cells CAPAN1 (48±6% specific lysis at 10:1 E:T ratio),but not PSCA negative 293T targets, and NT T cells produced onlybackground levels of lysis (7±4% and 4±1% specific lysis of CAPAN1 and293T cells, respectively).

Example 3 Targeting a Heterogeneous Tumor Using a Monospecific CAR-TCell Product Selects Immune Escape Variants

To determine whether CAR-PSCA T cell treatment could produce tumorelimination in vivo, SCID mice were engrafted with 1×10⁶ CAPAN1 cells (ahuman pancreatic cancer cell line), which naturally express PSCA, andwere modified with a γ-retroviral vector encoding eGFP-Fireflyluciferase (CAPAN1-eGFP-FFLuc) to allow for in vivo bioluminescencedetection. Once the tumor was established, as confirmed by an increasein the bioluminescence signal on two consecutive occasions, micereceived a single infusion of either NT or CAR-PSCA T cells (30×10⁶cells). As shown in FIG. 2 a CAR-PSCA T cell treatment resulted in aninitial anti-tumor response (day 28 post-treatment), this was followedby a rapid tumor progression which by days 42 and 56 post-treatment wassimilar between the two groups (FIG. 2 a, right panel).

To further investigate the reason for this immune escape, thisphenomenon was modeled in vitro by co-culturing NT or CAR-PSCA T cellswith CAPAN1-eGFP-FFLuc cells at a 5:1 ratio. After 72 hrs residualviable tumor cells were quantified by flow cytometry, gating on GFP+cells, while T cells were excluded by co-staining with a CD3-directedantibody. Similar to the in vivo findings, while co-culture of NT Tcells with the CAPAN1 cells had no impact on tumor cell growth, CAR-PSCAT cell treatment resulted in an initial anti-tumor response, reflectedby a 82±9% reduction in tumor cell numbers (FIG. 2 b). However, thetumor population that survived initial CAR-T cell exposure was resistantto re-treatment, as shown in FIG. 2 c, unlike tumor cells that wereoriginally treated with NT T cells, which retained their sensitivity toCAR-PSCA T cell treatment.

To determine the mechanism of resistance, IHC analysis was performed ontumor cells that had received a single treatment with either NT orCAR-PSCA T cells. As shown in FIG. 2 d, while CAR-PSCA T cellseliminated CAPAN1 cells expressing high levels of PSCA antigen, aresidual PSCA low/negative subpopulation remained, which subsequentlyoutgrew. These residual tumor cells did, however, continue to express asecond, untargeted, TAA, MUC1 (FIG. 2 d).

Example 4 Tumor Immune Escape Occurs Even Using T Cells Modified toExpress a 2^(nd) or 3^(rd) Generation CAR

Recent reports have shown that T cells modified to express CARscontaining co-stimulatory endodomains (2^(nd) and 3^(rd) generationCARs) have increased proliferation, cytokine production and prolonged invivo persistence. To test the hypothesis that more rapid or completekilling of cells expressing low levels of target antigen might preventemergence of tumor escape variants, and to discover whether tumor immuneescape could also be prevented using a later generation CAR construct, aCAR targeting PSCA was made that incorporated CD28 (2^(nd) generation)or CD28+41BB (3^(rd) generation) co-stimulatory endodomains. FIG. 7 ashows the 1^(st), 2^(nd) and 3^(rd) generation CAR-PSCA retroviralvector maps and FIG. 7 b shows expression of each in transduced primaryT cells. T cells expressing both 2^(nd) and 3^(rd) generation CARconstructs proliferated more than the 1^(st) generation construct whencultured with K562 cells modified to express PSCA antigen (FIG. 7 c),but all had equivalent cytolytic activity against CAPAN1 cells in bothshort-term (6 hr) ⁵¹Cr release (FIG. 7 d) and long term (72 hr)co-culture assays (FIG. 7 e), leaving behind the same residual resistanttumor subpopulation.

Example 5 T Cells Modified with a CAR Targeting MUC1 Specifically KillAntigen-Expressing Targets, but Tumor Heterogeneity Leads to TumorImmune Escape

Because heterogeneity of target antigen expression is an evident causeof tumor immune resistance and escape, it was next determined whetherconcomitant targeting of a second TAA, MUC1, would overcome thisproblem. A retroviral vector was generated encoding a CAR directedagainst MUC1, with a truncated CD19 molecule (ΔCD19) as a marker¹⁷. FIG.3 a shows the retroviral vector map. To test the activity of theMUC1-CAR T cells from 7 donors were transduced. FIG. 3 b (left panel)shows detailed results from one representative donor, and summarizeddata for all 7 donors. The mean CAR-MUC1 expression was 83.1% (±11.5%)(FIG. 3 b, right panel) and T cell phenotype was unaffected byCAR-transduction (FIG. 3 c). Again, the cultures consisted predominantlyof CD3+(95.2±5.7% and 97.2±2.0%) T cells, with CD4+(19.2±12.0% and12.3±8.1%) and CD8+(76.1±15.5% and 85.1±8.3%) subpopulations (NT andCAR-MUC1 T cells, respectively), which expressed similar levels of thecentral memory markers CD62L, CD27, and CD45RO. CD56+ CD3− NK cells werea mean of 1.7% (±3.0%) of NT and 2.2% (±2.3%) of CAR-MUC1 T cells.Transgenic CAR-MUC1 T cells could specifically kill CAPAN1 cells, whichnaturally express MUC1 antigen (35±5% specific lysis at 10:1 E:T ratio),and had no activity against 293T cells, which are MUC1 negative targets(FIG. 3 d).

To determine whether CAR-MUC1 T cell monotherapy would also lead totumor immune escape, SCID mice were again engrafted with the pancreaticcancer cells line CAPAN1-eGFP-FFLuc cells and treated with either NT orCAR-MUC1 T cells once the tumor was established. While CAR-T celltreatment produced an initial anti-tumor response measurable by adecrease in the tumor signal at day 28 post-treatment, this was followedby rapid tumor progression (FIG. 4 a). To confirm that this escape wasdue to tumor antigen modulation NT or CAR-MUC1 T cells were co-culturedwith CAPAN1-eGFP-FFLuc cells for 72 hrs and while CAR-MUC1 T celltreatment resulted in an initial reduction in tumor cells (66±21%) (FIG.4 b), those that remained proved insensitive to re-treatment (FIG. 4 c),likely due to decreased antigen expression, as confirmed by IHC (FIG. 4d). These residual tumor cells did, however, continue to express the TAAPSCA.

Example 6 Combination of Car-T Cells Targeting Two TAAs ProducesSuperior Anti-Tumor Activity

To determine whether dual-targeted CAR therapy would produce superioranti-tumor effects, the pancreatic cancer cells line CAPAN1 thatnaturally express both PSCA and MUC1, were cultured with both CAR-PSCAand CAR-MUC1 T cells simultaneously. In a short-term (6 hr) cytotoxicityassay, combination therapy produced superior tumor cell killing (75±8%specific lysis, E:T 10:1) compared to single antigen-specific T cells(35±5% and 48±6% specific lysis, E:T 10:1, CAR-MUC1 and CAR-PSCA,respectively) (FIG. 5 a). Similar results were obtained after a 3-dayco-culture, in which treatment with CAR-MUC1 T cells alone reduced tumorcells by 65±13%, treatment with CAR-PSCA T cells alone eliminated82.1±9% of tumor cells, while dual targeted therapy was superior,resulting in a 96.6±1% reduction (FIG. 5 b).

To address whether dual CAR-targeted therapy could result in tumorelimination, SCID mice were engrafted with CAPAN1-eGFP-FFluc cells. Asshown in FIG. 5 c and 5 d, treatment with a combination of CAR-MUC1 andCAR-PSCA T cells produced superior anti-tumor effects compared witheither tested individually. However, this effect was not sustained andby day 63 the tumor recurred in all animals. Thus, dual targeted therapywas also insufficient to eliminate all cancer cells.

Example 7 Generation of an Artificial System to Model Tumor ImmuneEscape

To better understand the mechanism behind this therapy failure, anengineered tumor model was developed by transgenically expressing eitherMUC1 or PSCA TAAs in 293T cells. Thus, two retroviral vectors weregenerated; the first encoding the MUC1 antigen and co-expressing thefluorescent tag mOrange and the second encoding the PSCA antigen andco-expressing GFP. The intensity of the fluorescent tag correlated withthe intensity of antigen expression, as shown in FIG. 8 for MUC1, thusallowing us to monitor, in real time, the anti-tumor activity of ourCAR-T cells. FIG. 6 a shows the retroviral vector maps and FIG. 6 bshows the expression of MUC1/mOrange and PSCA/GFP in 293T cells. Toensure that each tumor cell expressed a target antigen, these cells weresubsequently sorted to achieve pure populations that were either 100%MUC1-expressing (mOrange+) or PSCA-expressing (GFP+).

To mimic a heterogeneous tumor population the sorted cells were mixed ata 1:1 ratio and initially treated with either NT or CAR-PSCA T cells for72 hrs. NT T cell treatment had no impact on residual tumor cell numberswhereas treatment with CAR-PSCA T cells alone reduced the number ofengineered tumor cells by 50.9±1% (FIG. 6 c), reflecting a selectivereduction in the GFP+ (PSCA-expressing) population (98.1±1%). To examinethe kinetics of CAR-T cell killing tumor cell numbers were quantified,by flow cytometry, over time (0, 12, 24, 36, 48, and 60 hrs). As shownin FIG. 6 d, there was a progressive decrease in the number of GFP+tumor cells, which was most pronounced within the first 24 hrspost-treatment (45.4±11% reduction between 0-12 hrs and 29.7±4%reduction between 12-24 hrs), and less marked thereafter (10.7±4% from24-36 hrs, 10.2±1% from 36-48 hrs, and 1±0.9% from 48-60 hrs).Nevertheless, a residual PSCA-expressing subpopulation (1.9%) remained.To next investigate whether sensitivity to CAR-mediated killing waslinked to the intensity of target antigen expression on tumor cells, thefluorescence intensity of GFP was also measured at the same timepoints.As shown in FIG. 6 e, tumor cells expressing the highest antigen levelswere killed first, while those with lowest expression survived.

These studies were repeated but substituted with CAR-MUC1 T cells as theeffector population. After 72 hrs CAR-MUC1 T cell treatment the totalnumber of tumor cells had decreased by 49.9% (±5%) (FIG. 6 f),representing a selective and gradual reduction in MUC1/mOrange+ tumorcells over time (FIG. 6 g), until only MUC1 “low” tumor cells remained(FIG. 6 h), while the PSCA+ population was unaffected.

Next, to determine whether dual-targeted CAR therapy would producesuperior anti-tumor effects, the heterogeneous tumor cell population(1:1 mix of MUC1/mOrange+ and PSCA/GFP+ 293T cells) was co-cultured withboth CAR-MUC1 and CAR-PSCA T cells. However, even this strategy failedto eliminate all tumor cells and after 72 hrs 6.0±3% of cellsremained—3.9±2% residual MUC1/mOrange+ and 1.9±1% residual PSCA/GFP+cells (FIG. 6 i). Again, these residual subpopulations reflected cellswith the lowest intensity of target antigen expression, judged bymOrange and GFP fluorescence (FIGS. 6 j and 6 k). Thus, sensitivity toCAR-T cell treatment is related to both the proportion of cellsexpressing the targeted antigen as well as the intensity at which theantigen is expressed.

Finally, to determine whether CAR-T cell potency could be improved bycombination with conventional epigenetic modulators, which can increaseTAA expression by demethylating DNA, CAPAN1 cells were cultured that hadbeen previously treated with CAR-MUC1 T cells and consequently expressedonly low levels of the target antigen, with 1 μM of decitabine, ahypomethylating agent. Decitabine exposure resulted in an increase inthe intensity of MUC1 expression from 3.5 to 26.0 (relative meanfluorescence intensity) after 4 days of treatment (FIGS. 9 a and 9 b),re-sensitizing previously resistant tumor cells to CAR-MUC1 T cellkilling (FIG. 9 c).

Example 8 Combining Car T-Cells with Epigenetic Modifiers

FIG. 10 represents expression of CAR-MUC1 on cells that were selectedfor high expressing cells or low expressing cells.

FIGS. 11 and 12 provide a comparison of the effectiveness of effectorcells that have high or low expression of a CAR compared to tumor cellsthat express high or low levels of the antigen to which the CAR istargeted.

In FIG. 11, cells with either low expression of CAR-MUC1 or highexpression of CAR-MUC1 are examined for antitumor properties by assayingfor percentage of residual tumor cells that express the target antigenat a low level. Twenty four hours after exposure of the CAR-MUC1 T cellsto the tumor cells, those CAR-MUC1 T cells having high expression of theCAR are more effective than those CAR-MUC1 T cells having low expressionof the CAR in eliminating the tumor cells that have low expression ofthe MUC1 antigen.

In FIG. 12, there is an analogous assay in which cells with either lowexpression of CAR-MUC1 or high expression of CAR-MUC1 are examined forantitumor properties by assaying for percentage of residual tumor cellsfor tumor cells that express the target antigen at a high level. Twentyfour hours after exposure of the CAR-MUC1 T cells to the tumor cells,those CAR-MUC1 T cells having high expression of the CAR are moreeffective than those CAR-MUC1 T cells having low expression of the CARin eliminating the tumor cells that have high expression of the MUC1antigen.

However, when comparing FIG. 11 vs. FIG. 12, the difference is not sosignificant between the efficiency of the low-expressing andhigh-expressing CAR-MUC1 T cells in target cells that have highexpression of the target antigen. This indicates that the increase inexpression of CARs on effector cells has an effect particularly on tumorcells that have low expression of the target antigen.

FIG. 13 demonstrates that decitabine enhances CAR expression onmodified-T cells. It is illustrated therein that one can modulate theintensity of CAR-MUC1 expression on effector cells when the effectorcells are cultured with decitabine. After one day, and certainly afterseveral days, there is an increase in expression of the CAR-MUC1 on thecells.

FIG. 14 shows that decitabine enhances antitumor effects of CAR-MUC1T-cells. As illustrated, there is an antitumor effect when comparingCAR-MUC1 T cells alone compared to the control, which results in about80% tumor destruction. However, when the CAR-MUC1 T cells are combinedwith decitabine, there is increased potency by more than half than whatis seen with the CAR-MUC1 T cells alone. Therefore, combination ofdecitabine and CAR-MUC1 T-cells increased the antitumor effect.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

1.-62. (canceled)
 63. A method of enhancing potency of immune cells thatexpress at least one therapeutic protein, comprising contacting theimmune cells with an effective amount of a histone deacetylase (HDAC)inhibitor, and/or DNA methyl transferase (DNMT) inhibitor for a timesufficient for expression of said therapeutic protein to increase, ascompared to said immune cells not contacted with said respectivemitogen, HDAC inhibitor and/or said DNMT inhibitor, wherein said immunecells are T cells, NK cells, dendritic cells, or a mixture thereof. 64.The method of claim 63, wherein the HDAC inhibitor is trichostatin A,sodium phenylbutyrate, Buphenyl, Ammonaps, Valproic acid, Depakote,valproic acid, romidepsin (ISTODAX®), Vorinostat, Zolinza, panobinostat,belinostat, entinostat, JNJ-26481585, MGCD-010, or a combinationthereof.
 65. The method of claim 63, wherein said DNMT inhibitor is5-azacytidine, decitabine, zebularine, SGI-110, SGI-1036, RG108, caffeicacid purum, chlorogenic acid, epigallocatechin gallate, procainamidehydrochloride, MG98, or a combination thereof.
 66. The method of claim65, wherein said 5-azacytidine is VIDAZA®.
 67. The method of claim 63,wherein said immune cells are T cells.
 68. The method of claim 67,wherein said T cells are CD4+ T cells, CD8+ T cells, or Treg cells. 69.The method of claim 63, wherein said contacting is performed in vitro.70. The method of claim 63, wherein said contacting is performed in vivoin an individual comprising the immune cells.
 71. The method of claim63, wherein said immune cells and said HDAC inhibitor or said DNMTinhibitor are administered to said individual separately.
 72. The methodof claim 63, wherein said HDAC inhibitor and/or DNMT inhibitor, and saidimmune cells, are administered to the individual in separatepharmaceutical formulations.
 73. The method of claim 63, wherein saidHDAC inhibitor and/or DNMT inhibitor, and said immune cells, areadministered to the individual in the same pharmaceutical formulation.74. The method of claim 63, wherein said immune cells are contacted withat least one HDAC inhibitor and at least one DNMT inhibitor.
 75. Themethod of claim 74, wherein said immune cells are contacted with saidHDAC inhibitor in vitro, and subsequently contacted with said DNMTinhibitor in vivo.
 76. The method of claim 74, wherein said immune cellsare contacted with said DNMT inhibitor in vitro, and subsequentlycontacted with said HDAC inhibitor in vivo.
 77. The method of claim 63,wherein expression of said therapeutic protein in said immune cells iscontrolled by a promoter repressor region, at least a portion of thesequence of which is methylated, and wherein said methylation results inenhanced expression of said therapeutic protein.
 78. The method of claim63, wherein said therapeutic protein is a chimeric antigen receptor(CAR).
 79. The method of claim 78, wherein the CAR comprises at leastone extracellular antigen-binding domain and at least one intracellularsignaling domain.
 80. The method of claim 63, wherein said immune cellsare autologous to a recipient of said immune cells.
 81. The method ofclaim 63, wherein said immune cells are allogeneic to a recipient ofsaid immune cells.
 82. The method of claim 63, wherein the immune cellscomprise two or more different therapeutic proteins.